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Improved recovery of protein from soy grit by enzyme-assisted alkaline extraction
Journal of Food Engineering 276 (2020) 109894
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Journal of Food Engineering journal homepage: http://www.elsevier.com/locate/jfoodeng
Improved recovery of protein from soy grit by enzyme-assisted alkaline extraction Milica N. Perovi�c a, Zorica D. Kne�zevi�c Jugovi�c b, Mirjana G. Antov a, * a b
University of Novi Sad, Faculty of Technology, Blvd. Cara Lazara 1, Novi Sad, Serbia University of Belgrade, Faculty of Technology and Metallurgy, Karnegijeva 4, Belgrade, Serbia
A R T I C L E I N F O
A B S T R A C T
Keywords: Protein Extraction Carbohydrases Alkali Functional properties Soy grit
Recovery of protein from soy grit, its functional properties and possibility for the reduction of time of conven tional alkaline extraction by the assistance of enzymes were studied. Enzymatic treatment was performed by commercial preparations of cellulase (NS22086), xylanase (NS22083) and pectinase (Vinozym) (applied sepa rately or in combination) as well as by commercial carbohydrases cocktail (Enzyme complex, NS22119). Three different extractions were investigated - alkaline (at pH 8 for 1 h, 2 h or 3 h), enzyme-assisted aqueous (at pH 5.5 for 3 h) and enzyme-assisted alkaline extractions (enzymatic extraction for 1 h followed by alkaline extraction for 1 h or 2 h) at 50 � C and solid:liquid ratio 1:10 (w/v). The highest enhancement of recovery of protein was achieved by pretreatment of soy grit with enzyme cocktails. Treatment with Enzyme complex followed by 1 h alkaline extraction increased protein yield for 21% compared to 2 h alkaline extraction. Treatment by combi nation of individual cellulase, xylanase and pectinase followed by 2 h alkaline extraction enhanced protein yield for 13% in comparison to 3 h alkaline extraction. So, reduced time of alkaline extraction was attained by the assistance of carbohydrases cocktails with even positive effect on protein yield. In addition, protein from enzymeassisted alkaline extraction exhibited ameliorated solubility, emulsifying and whipping properties compared to alkaline extracted protein.
1. Introduction
sources such as extraction with aqueous and organic solvents (solutions with alkali, acid, salt, phenols, various buffers) that can be ultrasound, and enzyme-aided. Among all these methods, alkaline extraction is the most commonly used technique for protein extraction from plant and seed sources. Mechanism of alkali used for protein extraction from plants occurs through disrupting the cell wall by partial removal of lignin and by altering chemical composition and structure of (hemi) cellulose; in addition, the influence of alkali on protein extractability conducts also by its increased solubility (Sari et al., 2015). Great ad vantages of this traditional extraction method reflect in its simplicity, rapidity and low cost. Conventionally, soy protein isolates and concen trates are extracted from defatted soy flours using dilute alkali solutions (pH 8–9) at elevated temperature, followed with protein precipitation and concentration into a curd. Soy protein isolate yields are between 30 and 40% based on the defatted soy flakes/flour weight or approximately 60% of the protein in the flakes (Mounts et al., 1987). However, a traditional alkaline process can bring a few disadvantages as protein properties are changed along the extraction process. These changes include denaturation, racemization and formation of dehydro and
The importance of food proteins is well established - besides repre senting a source of energy and essential nutrients, these proteins fulfill another important role as they confer physicochemical characteristics that affect sensory properties and quality of food. Therefore proteins are considered the most valuable functional ingredients in food production. Over the last few decades growing global demand for food proteins has driven the search for new, sustainable protein sources based on plants able to potentially complement or replace animal proteins in various food applications (Rommi, 2016). Among them, soy occupies a promi nent place because soy protein is a complete protein that contains all essential amino acids required for normal human growth and develop ment (Singh et al., 2008). Nowadays, a consumption of soy products has increased significantly and the soybean protein is highly valued for its nutritional quality and functional properties. Soy food comes in dozens of forms including flour, protein isolates and concentrates, infant for €her et al., 2011). mulas and textured fibers (Stro A number of methods are used for the recovery of proteins from plant * Corresponding author. E-mail address: [email protected] (M.G. Antov).
https://doi.org/10.1016/j.jfoodeng.2019.109894 Received 10 June 2019; Received in revised form 20 December 2019; Accepted 22 December 2019 Available online 26 December 2019 0260-8774/© 2019 Elsevier Ltd. All rights reserved.
M.N. Perovi�c et al.
Journal of Food Engineering 276 (2020) 109894
cross-linked amino acids, reflected through poor solubility, lower nutritional quality and functional characteristics of extracted protein (Friedman, 1999; Sari et al., 2015). It was shown that these protein damaging processes were enhanced at higher pH and temperature and also increased with the increase in alkaline extraction time (Friedman, 1999; Schwass and Finley, 1984). Another approach to help disintegration of the plant cell wall that acts as a barrier for diffusion of protein into solvent, and to consequently enhance protein extractability, is the degradation of structural poly saccharides by the use of carbohydrases (Rosset et al., 2014). Generally, enzyme-assisted extractions are recognized as eco-friendly technologies as they offer a possibility of greener chemistry in a search for cleaner routes in food industry. Recent studies on enzyme-assisted extraction have shown faster extraction, higher recovery, reduced solvent usage and lower energy consumption when compared to non-enzymatic methods thus representing a potential alternative to conventional sol vent based extraction methods (Puri et al., 2012; Vergara-Barberan et al.,2015; Ribeiro Garcia De Figueiredo et al., 2018; V� asquez et al., 2019). Cell wall degrading carbohydrases - cellulase, pectinases and hemi cellulases are enzymes that hydrolyze plant cell wall components which leads to disruption of structural integrity and consequent increase in permeability of cell wall, finally resulting in enhancement of the extraction yield (Puri et al., 2012). Enzymes are usually used for the treatment of plant material prior to conventional methods of extraction. Some studies on enzymes-assisted extractions of soy flour showed promising results regarding improved yield of protein (Jung et al., 2006) and oil (Rosenthal et al., 2001) as well as nutritional and sensory properties of prepared extract (Wei et al., 2018). However, some of them reported no beneficial effect (Ouhida et al., 2002; Rosset et al., 2014) or even, under certain conditions, slightly decreasing effect (Rosenthal et al., 2001) on protein yield upon enzymatic treatment of soy flour. The aim of this study was to investigate the efficacy of cell wall degrading enzymes – cellulase, pectinase and xylanase, applied as single activity or in combination, in extraction of protein from defatted soy grit. The contents of cellulose, hemicellulose and pectin in soy flour/grit � and Zralý, cover a broad range of values (Jung et al., 2006; Písa�ríkova 2010; Rosenthal et al., 1998), therefore, the usage of enzymes that lead to increased protein extraction need to be established. The combination of enzymatic and alkaline extraction of protein with the aim of abridg ment of alkaline extraction by enzymatic pretreatment of soy grit was also investigated. In addition, functional characteristics of protein ob tained by enzymatic-alkaline extraction, such as solubility, emulsifying and whipping characteristics, important for its behaviour in food sys tems, were determined and compared to those of protein extracted by alkali.
Process, Novozymes) activity were used for enzymatically enhanced extraction of soy protein. In addition, one commercial cocktail of wide range of carbohydrase – cellulase, xylanase, pectinase, arabinanase and β-glucanase (Enzyme complex, NS22119, Novozymes’ Bioethanol Kit) was also applied for enzymatic treatment of soy grit. Cellulolytic activity of Cellulase complex and Enzyme complex assayed according to Ghose (1987) was determined to be 340 FPU/mL and 30 FPU/mL, respectively. One FPU was defined as the amount of enzyme that released 2 mg of reducing sugars from 50 mg of Whatman No.1 filter paper in 1 h under the standard conditions of hydrolysis (temperature 50 � C, pH 4.8). Xylanolytic activity of xylanase preparation NS22083 and Enzyme complex assayed according to Bailey et al. (1992) was determined to be 375 U/mL and 6 U/mL, respectively. One unit (U) of xylanase activity was defined as the amount of enzyme that liberates 1 mmol of xylose per minute under the assay conditions (temperature 50 � C, pH 5.3). Pectinolytic activity of Vinozym and Enzyme complex assayed ac cording to Patil and Dayanand (2006) was determined to be 7.8 U/mL and 1.1 U/mL, respectively. One unit (U) of pectinase activity was defined as the amount of enzyme that catalyzes liberation of 1 mmol of galacturonic acid per min at defined conditions of assay (temperature 45 � C, pH 4.5). 2.3. Extraction of protein from soy grit 2.3.1. Alkaline extraction Alkaline extraction of soy protein was carried out by distilled water with pH adjusted to 8 by NaOH, at solid:liquid ratio 1:10 (w/v) for 1 h, 2 h or 3 h, at 50 � C and constant stirring. After the extraction was completed, the suspension was centrifuged at 10,000 rpm for 15 min (Sorvall, RC-5B). Protein concentration in the supernatant was measured according to Lowry method (Lowry et al., 1951). 2.3.2. Enzymatic extractions In order to evaluate the effect of individual enzyme activities, extraction of soy protein was performed using single enzyme prepara tion in following dosages per gram of dry matter: 20 FPU cellulase from NS22086, 33 U xylanase from NS 22083 or 0.4 U pectinase from Vino zym. In addition, the effect of commercial enzyme cocktail (NS22119) and combination of individual enzyme preparation (NS22086, NS22083, Vinozym) named as Mix on recovery of protein was also evaluated. NS22119 was added at dosage corresponding to cellulolytic activity 20 FPU/g DM (followed by 4 U xylanase and 0.7 U pectinase per g DM), while carbohydrases Mix contained all three single enzyme dosages equal to those used in experiments with individual activities. Enzyme-assisted aqueous extractions were carried out under the same conditions as alkaline extraction - at 50 � C and solid:liquid ratio 1:10 (w/v) for 3 h but pH was 5.5. This pH fitted within the optimal pH ranges for all used enzyme activities (Novozymes, 2010). During the extraction, pH was controlled and adjusted to initial value with NaOH and HCl solutions. In order to inactivate enzymes after completed extraction, suspensions were boiled for 5 min at 100 � C. Control run was performed using same treatment conditions without enzymes. Combined enzymatic and alkaline extractions were performed by applying enzymatic treatment of defatted soy grit under the same con ditions described above for 1 h followed by alkaline extraction for 1 h or 2 h. After completed extraction processes, suspensions were cen trifugated at 10,000 rpm for 15 min (Sorval RC-5B) and protein con centration in supernatants was measured by Lowry method. Control was conducted at the same conditions described above but the part that corresponded to the enzymatic treatment was performed without enzymes.
2. Materials and methods 2.1. Soy grit Toasted soy grit, defatted by hexane extraction, was a gift from Sojaprotein (Be�cej, Serbia). Dry matter (DM) content determined by oven drying at 60 � C until constant mass was 98.7%. Protein content of defatted soy grit was determined by Kjeldahl (Official Methods of Analysis, 1995). Total dietary fibers (TDF), insoluble dietary fibers (IDF) and soluble dietary fibers (SDF) in defatted soy grit were determined using Total Dietary Fiber Assay Kit (Megazyme, Ireland). Before and during experimental analyses soy grit was stored in the refrigerator at constant temperature (4 � C) monitored by thermometer. 2.2. Commercial enzyme preparations
2.4. Protein precipitation and drying
Three commercial enzyme preparations having principally cellulo lytic (Cellulase complex, NS22086, Novozymes’ Bioethanol Kit), xyla nolytic (NS22083, Novozymes’ Bioethanol Kit) or pectinolytic (Vinozym
Before 2
electrophoresis
and
determination
of
functional
M.N. Perovi�c et al.
Journal of Food Engineering 276 (2020) 109894
characteristics, extracts of soy protein were concentrated and dried. Knowing that isoelectric point of soy protein is at pH 4.5 (Jiang et al., 2010), 1M HCl was used to adjust pH in supernatants and precipitate extracted proteins. After precipitation, protein curds were collected by centrifugation (10,000 rpm, 10 min) and resolved in distilled water at pH 7. Obtained protein solutions were subjected to freeze drying at - 40 � C for 24 h at laboratory lyophilisation equipment (Martin Crist Alpha LSC 2–4, Osterode, Germany).
Table 1 Content of protein and dietary fibers in defatted soy grit. Component
Content (% DM)
Protein TDF IDF SDF
46.16 � 0.00 34.42 � 0.25 27.18 � 0.03 5.58 � 0.35
TDF ¼ total dietary fiber, IDF ¼ insoluble dietary fiber, SDF ¼ soluble dietary fiber.
2.5. Electrophoresis Samples from alkaline and combined enzymatic and alkaline ex tractions were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% TruPAGE Precast Gels (SIGMAALDRICH, Taufkirchen, Germany) under reducing conditions with discontinuous buffer system (Laemmli, 1970). Ten-μL samples (protein concentration 2 mg/mL) were loaded onto each well and electrophoresis was run at 180 V and 100 mA for 45 min. Gels were fixed and stained with Coomassie Blue R250 for 1 h and de-stained in distilled water, and the relative molecular weights of the protein fractions were compared with those of marker proteins (SERVA Electrophoresis, Heidelberg, Germany).
FSð%Þ ¼
3. Results and discussion In most grain legumes, which are used in human nutrition as valu able sources of protein, the content of cell wall materials i.e. dietary fiber ranges from 8% to 27.5% and differs among species and the vari eties; however, the content of dietary fiber in legume seed also varies according to their processing conditions (Guillon and Champ, 2002). Dietary fiber (DF) can be defined as carbohydrate polymers with 10 or more monomeric units which are not hydrolyzed by the endogenous enzymes in the small intestine of humans (CODEX Alimentarius Com mission, 2010). Dietary fiber consists of a variety of non-starch poly saccharides which include cellulose, hemicellulose, pectin, β-glucans and lignin. Hemicellulose, cellulose and lignin bind only little water and they are characterized as insoluble dietary fibers (IDF). On the other side, pectin substances and β-glucans are termed as soluble dietary fibers (SDF) because of their ability of binding water and swell. Defatted soy grit used in this study was analysed for protein and dietary fiber content and the results expressed as percentages of dry matter (DM) are pre sented in Table 1. Results showed that protein represented almost half of dry weight of defatted soy grit which generally, though somewhat higher, was in accordance with results reported in literature (Erickson, 2016; Rosen thal et al., 1998). Approximately one third of material dry weight was composed of cell wall components with dominant fraction of insoluble dietary fiber which can be attributed to high cellulose and hemicellulose content while soluble fiber related to pectin represented smaller portion. It is known that content of dietary fiber in soy flour/grit might cover a broad range of values. Literature data for TDF, IDF and SDF contents in different soybean genotypes ranged from 20.34 to 38.4 g, 18.19–35.4 g and 1.57–9.7 g per 100 g, respectively (Salmani et al., 2012; Pascoal et al., 2015; Písa�ríkov� a and Zralý, 2010). Determined contents of TDF, IDF and SDF in defatted soy grit used in this study were in good agreement with values reported previously.
(1)
Emulsion oil in water was prepared by mixing protein solution and sunflower oil in ratio 85:15 (v/v). Protein solutions were prepared by dissolving extracted protein in 0.3 M NaCl at pH 8 to make different concentrations – 0.2, 0.3 or 0.5 g/100 mL. Sodium azide was added as antimicrobial agent in concentration 0.02% (w/v). Oil and continuous water phase were homogenized with ULTRA-TURRAX (T-25, IKA) at 15,000 rpm for 10 min at 25 � C. Emulsion stability was evaluated by creaming test - immediately after preparation, emulsions were trans ferred into 10 mL graduated glass cylinders (VE); during the time emulsions separated into emulsion serum layer (VS) and emulsion cream; volume of serum layer was measured at room temperature and creaming index (CI) was calculated as: VS 100 VE
(2)
In addition, whipping ability of protein samples was investigated by determining foam capacity and foam stability. Protein solution (0.5 g/ 100 mL, 0.3 M NaCl, pH 8) was whipped at 15,000 rpm for 10 min at 25 � C. After whipping, the propeller was immediately removed and the glass cylinder sealed with parafilm to avoid the foam disruption. Vol umes were measured in starting minute (VO) and at 4th (V4) and 30th (V30) minute.Foam capacity (FC) was calculated as: FCð%Þ ¼
V4
V0 V0
100
(4)
Statistical analysis were carried out using Statistica (TIBCO Softver Inc.) to compare differences between results by the analysis of variance (One-way ANOVA), followed by Tukey’s HSD test. Results of statistical analysis were assumed to be significant when p < 0.05, very significant when p < 0.01 and highly significant when p < 0.001. Each experiment was carried out in triplicate and the results were presented herein as mean value � standard deviation.
2.7. Emulsifying and whipping characteristics
CIð%Þ ¼
100
2.8. Statistical analysis
Protein sample was dissolved in 0.3 M NaCl (Kinsella, 1979) at pH 8 to final concentration C1 of 4 g/L and thoroughly mixed on shaker at room temperature for 1 h after which pH was controlled and adjusted to 8 if necessary. Suspension was left overnight at 4 � C for better protein hydration and unsolved particles were separated by centrifugation at 10, 000 rpm for 10 min. Concentration of dissolved proteins in the super natant (C2), measured by Lowry method, was used to calculate solubility (S) according to Eq. (1): C2 100 C1
V0 V0
Microphotographs of prepared emulsions were taken on an optical microscope, Biooptica BEL3000, Germany at 40x magnification.
2.6. Solubility
Sð%Þ ¼
V30
3.1. Alkaline extraction
(3)
Defatted soy grit was extracted by alkaline aqueous solution (pH 8) at 50 � C for 1 h, 2 h or 3 h under constant stirring and after centrifu gation protein concentration in the supernatant was determined.
while foam stability (FS) was calculated according to Eq. (4): 3
M.N. Perovi�c et al.
Journal of Food Engineering 276 (2020) 109894
3.2. Enzymatic extraction of protein Results presented in Table 1 showed that dietary fiber occupied almost one third of raw material dry weight, therefore in the next ex periments the effect of carbohydrases (individual or in combination) on the extraction of protein from soy grit was investigated. Dosages of in dividual activities were previously determined as those ones enabling the highest protein yield under applied conditions (data not shown). After solid/liquid separation, released protein was determined and re sults are presented in Fig. 2, along with results of the control extraction without enzymes. Action of cellulase, pectinase and Enzyme complex during aqueous extraction of defatted soy grit increased protein yield with high signif icance (p < 0.001) in comparison to control, while the addition of car bohydrases Mix increased it significantly (p ¼ 0.0364). However, when individual xylanase was added increase in protein yield was not signif icant (p ¼ 0.5265). Regarding the effect of individual enzyme activities, the most distinguished was that of cellulase (p ¼ 0.0001) with the protein yield of 27.56 � 1.24%, which can be explained by the high IDF i.e. cellulose content in soy grit. The highest protein yield 34.97 � 2.02% was achieved by commercial Enzyme complex representing highly sig nificant increase (p ¼ 0.0001) compared to control; this cocktail con tained wide range of carbohydrases including cellulase, pectinase and xylanase in the most adequate portions for the efficient joint hydrolysis of cell wall matrix and release of protein. The result of joint enzyme action within the Enzyme complex as the most efficient in the enhancement of protein yield was in agreement with recent model od plant cell wall as the single network composed from joined all structural polysaccharides (Dick-P�erez et al., 2011). In addition, it was shown that enzymatic degradation of one structural polysaccharide (cellulose) was significantly facilitated after removal of other cell wall components (Ouhida et al., 2002). It is worth to notice that protein yield reached by the use of commercial Enzyme complex was for 6% higher than that after 1 h alkaline extraction. However, comparing to the results of 3 h alkaline extraction protein yield from enzymatic aqueous extractions was lower from 16% to 57%. This can be explained by pH 5.5 of the
Fig. 1. Yield of protein from defatted soy grit obtained by alkaline extraction; conditions: pH 8, temperature 50 � C, extraction time 1 h, 2 h or 3 h. Different letters represent the differences in means of the samples, according to Tukey’s HSD test at p < 0.05 level.
Extraction yield of protein expressed as percentage of soy grit dry matter is presented in Fig. 1. Yield of protein significantly increased (p < 0.05) with increasing the time of alkaline extraction within investigated period. After 2 h extraction protein yield, 37.84 � 0.57%, was very significantly improved (p ¼ 0.0058) for 15% compared to yield from 1 h extraction. Further prolongation to 3 h extraction enabled achievement of protein yield 41.03 � 1.96% which represented significant increase compared to 2 h extraction (p ¼ 0.0398). This result was in accordance with yield of protein extracted from defatted soy flour by alkali under similar con ditions (Mounts et al., 1987; Sari et al., 2015).
Fig. 2. Yield of protein from defatted soy grit ob tained by enzyme-assisted aqueous extractions; con ditions: pH 5.5, temperature 50 � C, time: 3 h; enzyme dosages/g DM: 0.4 U pectinase (Vinozym), 33 U xylanase (NS22083); 20 FPU cellulase (NS22086); E. complex - 0.7 U pectinase, 4 U xylanase and 20 FPU cellulase; MIX - 0.4 U pectinase (from Vinozym), 33 U xylanase (from NS22083) and 20 FPU cellulase (from NS22086); control without enzymes. Different letters represent the differences in means of the samples, according to Tukey’s HSD test at p < 0.05 level.
Enzymatic extraction
50 45
a
Extraction yield (% DM)
40 35
b
b
30 25 20
c
c,d
d
15 10 5 0
control
pectinase
xylanase
cellulase
E complex 4
MIX
M.N. Perovi�c et al.
Journal of Food Engineering 276 (2020) 109894
Fig. 3. Yield of protein from defatted soy grit ob tained by enzyme-assisted alkaline and alkaline ex tractions: a) 1 h at pH 5.5 followed by 1 h pH 8, and b) 1 h at pH 5.5 followed by 2 h at pH 8; temperature: 50 � C; enzymes dosages/g DM: 0.4 U pectinase (Vinozym), 33 U xylanase (NS22083); 20 FPU cellu lase (NS22086); E. complex - 0.7 U pectinase, 4 U xylanase and 20 FPU cellulase; MIX - 0.4 U pectinase (from Vinozym), 33 U xylanase (from NS22083) and 20 FPU cellulase (from NS22086); controls: 1 h at pH 5.5 without enzymes followed by 1 h or 2 h at pH 8 and 50 � C. Different letters represent the differences in means of the samples, according to Tukey’s HSD test at p < 0.05 level.
a)
b)
extraction experiments, which was appropriate for the enzyme action but did not enable maximal protein solubility as discussed previously (Sari et al., 2015). Presented protein yield corresponded to this extracted under similar experimental conditions from soy flakes (Jung et al., 2006).
regardless Enzyme complex (increase for 38%) or carbohydrases Mix (increase for 22%) was used for enzymatic treatment followed by 1 h alkaline extraction (Fig. 3a). When treatment with Enzyme complex and Mix was followed by 2 h alkaline extraction protein yield was increased for 31% and 28%, respectively, which also represented highly signifi cant improvement (p ¼ 0.0002) compared to corresponding control (Fig. 3b). Moreover, after enzymatic treatment with carbohydrases Mix and Enzyme complex followed by 1 h alkaline extraction protein yield, 40.87 � 1.37% and 45.93%, respectively, was almost equal or higher than this after 3 h alkaline extraction (41.03 � 1.97%). On the other side, statistical analysis showed that the enhancement of protein yield by treatment with individual enzymes was significant only when it was followed by 1 h alkaline extraction. Observed enhancement was at very significant level (p ¼ 0.0014) and significant level (p ¼ 0.0234) for xylanase and pectinase, respectively. So, results showed that under applied conditions treatment with enzyme cocktails enhanced protein extraction more markedly than that with individual enzymes which was contrary to findings of Jung et al. (2006). Nevetheless, in comparison to 2 h alkaline extraction, protein yields
3.3. Enzyme-assisted alkaline extraction Combination of enzyme-assisted aqueous extraction and alkaline extraction was performed primarily to reduce alkaline extraction time in order to decrease possible protein damage and formation of undesired products. Moreover, this combined extraction process should overcome restraints of enzyme-assisted aqueous extraction regarding the effect of applied pH on protein solubility. Two combined extractions included 1 h enzymatic treatment followed by 1 h or 2 h alkaline extraction, and results of protein yield as the first response are presented in Fig. 3. In two and 3 h combined extractions the most prominent increase of protein yield in comparison to corresponding controls was achieved with enzyme cocktails. This increase was highly significant (p ¼ 0.0002) 5
M.N. Perovi�c et al.
Journal of Food Engineering 276 (2020) 109894
Table 2 Functional characteristics of protein from defatted soy grit extracted by alkaline and carbohydrases MIX-assisted alkaline extraction. Extractiona
Solubility (%)
Foam capacity (%)
Foam stability (%)
Alkaline
73.67 � 1.01a 79.90 � 0.80b
57.50 � 3.53a
49,00 � 1.41a
107,00 � 4.24b
89.73 � 0.37b
Carbohydrases MIXassisted a
Alkaline: 3 h at pH 8 and 50 � C; carbohydrases MIX-assisted: 0.4 U pectinase, 33 U xylanase and 20 FPU cellulase activity/g DM, 1 h at pH 5.5 followed by 1 h alkaline extraction at pH 8, 50 � C. Different letters represent the differences in means of the samples, according to Tukey’s HSD test at level p < 0.001 for solubility and foam stability, and at level p < 0.01 for foam capacity.
order to perceive the effect of applied extraction conditions. Analysed protein samples originated from extractions yielding approximately equal amounts of protein. 3.5.1. Solubility It is well known that solubility of food proteins is one of most valu able functional properties in general. Typically, higher protein solubility correlates with good gelling, foaming and emulsifying properties (Joo kyeing, 2011). Results of solubility of the investigated protein samples at pH 8 are presented in Table 2. The higher solubility, 79.90 � 0.80%, was determined for protein extracted by combined treatment with carbo hydrase MIX; though lower than literature data for soy protein isolate (Rickert et al., 2004) this represented highly significant improvement (p ¼ 0.0004) of solubility in comparison to sample from alkaline extraction. It is known that protein properties are affected by the processing treatments and environment (Kinsella, 1979) and extraction conditions that include heat and alkaline treatment can be related to denaturation leading to lower solubility (Sari et al., 2015). Reason for the enhanced solubility of protein from enzyme-assisted alkaline extraction can be related with lower degradation of protein during extraction process due to reduced duration of protein exposure to alkali.
Fig. 4. SDS–PAGE of proteins from pectinase-assisted followed by 2 h alkaline (line 1), 3 h alkaline (line 2) and carbohydrase MIX-assisted followed by 2 h alkaline (line 3) extractions, loaded by 20 μg each; protein molecular weight markers are in lane M and corresponding molecular weights (in kDa) are indicated at side.
after treatment with individual enzymes followed by 1 h alkaline extraction had mutually very close values. Statistical analysis of the results obtained from enzyme-assisted al kali extractions showed that prolongation of alkaline part of extraction was not justified for all enzymatic treatments (individual or in combi nation). Judging by the significance of the enhancement, 2 h compared to 1 h alkaline extraction after enzymatic treatment improved protein extractability only when cellulase (significantly, p ¼ 0.012) and carbo hydrases Mix (highly significantly, p ¼ 0.0008) were used. With respect to protein yield presented results showed that by enzymatic treatment of soy grit particularly with enzyme cocktails reduction of time of alkaline extraction can be accomplished, with no decreasing but even with the enhancing effect.
3.5.2. Emulsifying characteristics The ability of protein to help the formation and stabilization of emulsions is very important for many food applications. The stability of emulsions prepared with protein samples from alkaline and carbohy drases Mix-assisted extractions was evaluated by monitoring of the creaming index in emulsions prepared with 15% oil and values of this indicator are shown in Fig. 5. Results revealed that increase in protein concentration of both tested samples increased the stability of prepared emulsions and that for both of them final values of the creaming index were similar. Comparing stabilities of emulsions prepared with equal concentration of protein extracted by different procedures, differences between them increased also with increasing protein concentration. As a result, the highest applied concentration of protein from enzyme-assisted alkaline extrac tion prolonged time needed for emulsion separation for approx. 10 min in comparison with alkali extracted protein under applied conditions thus indicating its improved emulsifying activity regarding stabilizing effect on oil-in-water emulsions. It is known that surface activity of protein that is related to their ability to lower the interfacial tension between water and oil is closely correlated with solubility and flexibility of protein molecule (Kinsella, 1979). In addition, it was shown that although denatured soy protein expressed higher interfacial pressure than native, emulsions prepared with native sample were more stable (Palazolo et al., 2003). So, presented results can be explained by milder extraction conditions in combined extraction comprised from enzymatic treatment and reduced exposure to alkali, which enabled higher solu bility and possibly higher ability for structure restoration (i.e. flexibility) of protein leading to improved stabilizing effect on emulsions. This
3.4. SDS-PAGE Samples from alkaline as well as from enzyme-assisted alkaline extraction were subjected to electrophoresis to evaluate pattern of extracted protein. After isoelectric precipitation and freeze-drying, samples of soy protein obtained by alkaline and two combined extrac tions were loaded in equal protein amounts. Proteins extracted by combination of enzymatic treatment with carbohydrases MIX and Vinozym followed by 2 h alkaline extraction were chosen based on the yield higher and lower than this from alkaline extraction, respectively. Results from SDS-PAGE (Fig. 4) showed that protein pattern in all ana lysed samples was unique indicating the presence of subunits of major soy storage proteins with approximate molecular weights 80, 75, 50, 40 and 20 kDa (Krishnan et al., 2009; Rovaris et al., 2013; Thanh and Shibasaki, 1977) as well as around 17 kDa band corresponding to Kunitz ~o �n, 1995). This revealed that the trypsin inhibitor (Petruccelli and An same protein fractions were extracted by alkaline and enzyme-assisted alkaline extractions indicating that differences in yields can be attrib uted to variation in degree of cell wall destruction which led to differ ences in protein release. 3.5. Functional characteristics of extracted protein Some functional characteristics of protein samples from alkaline and enzyme-assisted alkaline extractions were determined and compared in 6
M.N. Perovi�c et al.
Journal of Food Engineering 276 (2020) 109894
Fig. 5. Creaming index of emulsions prepared with protein from a) 3 h alkaline and b) carbohydrases MIX-assisted alkaline extractions.
result is also in an agreement with higher solubility expressed by protein originated from enzyme-assisted alkaline in comparison to that from alkaline extraction. Emulsifying ability of protein samples was also perceived through analysis of images of emulsions previously tested for stability (Fig. 6).
Practically, images confirmed previous finding based on the measure ment of creaming index considering the fact that emulsions with smaller droplets size are generally more stable. It was visible that protein from both samples at higher concentrations produced smaller oil drops; generally, it is known that higher protein concentration is able to better 7
M.N. Perovi�c et al.
Journal of Food Engineering 276 (2020) 109894
Protein concentration (%, w/v)
Extraction
0.2
0.3
0.5
Alkaline
MIX-assisted alkaline
Fig. 6. Images of oil droplets from emulsions with different concentrations of protein from alkaline and carbohydrases MIX-assisted alkaline extractions.
cover surface area of oil drops and to stop progressive coalescence of oil droplets and oil separation (Palazolo et al., 2003). Furthermore, it was also visible that protein from enzyme-assisted alkaline extraction pro duced relatively smaller oil droplets. Another property of protein that is important for some food appli cations is the capacity to form stable foams with gas by forming impervious protein films. Whipping ability of two protein samples was evaluated by measuring foam capacity and stability (Table 2). Higher foam capacity and stability, 57.5 � 3.53 and 107 � 4.24%, respectively were determined with protein from carbohydrases Mix-assisted alkaline in comparison to that from alkaline extraction (49 � 1.41 and 89.73 � 0.37%, respectively). This improvement was at level very significant (p ¼ 0.0064) and highly significant (p ¼ 0.0009) for foam capacity and foam stability, respectively. Good whipping characteristics of proteins are usually related to their good solubility in disperse (aqueous) phase but also to greater exposure of hydrophobic regions caused by dena turation (Kinsella, 1979; Van Vliet et al., 2002). However, it was also shown that soy protein exhibited superior foaming properties when it was native and soluble (Yasumatsu et al., 2014). Our results showed that reduced time of the exposure to alkali by previous enzymatic treatment favored extraction of protein with improved whipping characteristics indicating its increased ability of making film at the interface, which was in agreement with previously discussed results of its higher solubility.
treatment with commercial Enzyme complex and carbohydrases Mix (cellulase, xylanase and pectinase) followed by alkaline extraction. Combination of enzymes appeared to be the most adequate for the efficient hydrolysis of cell wall matrix and release of protein. Beside the positive effect on protein yield, functional characteristics of protein (solubility, emulsifying and whipping properties) from enzyme-assisted alkaline extraction were advanced compared to those from alkaline extraction. Presented results allowed reduction of time of alkaline extraction by its combination with enzymatic treatment, which posi tively affected both protein yield and functional properties as desirable attributes for protein application in wide range of food products. CRediT authorship contribution statement Milica N. Perovic�: Formal analysis, Conceptualization, Writing original draft. Zorica D. Kne� zevi� c Jugovi� c: Project administration, Validation. Mirjana G. Antov: Conceptualization, Supervision, Writing - review & editing. Acknowledgement The financial support from EUREKA Project SOYZYME (Grant No E! 9936) is greatly acknowledged. The authors also acknowledge Novo zymes for kindly gifted us by Novozymes Cellulosic Ethanol Enzyme Kit.
4. Conclusion
References
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Contents lists available at ScienceDirect
Journal of Food Engineering journal homepage: http://www.elsevier.com/locate/jfoodeng
Improved recovery of protein from soy grit by enzyme-assisted alkaline extraction Milica N. Perovi�c a, Zorica D. Kne�zevi�c Jugovi�c b, Mirjana G. Antov a, * a b
University of Novi Sad, Faculty of Technology, Blvd. Cara Lazara 1, Novi Sad, Serbia University of Belgrade, Faculty of Technology and Metallurgy, Karnegijeva 4, Belgrade, Serbia
A R T I C L E I N F O
A B S T R A C T
Keywords: Protein Extraction Carbohydrases Alkali Functional properties Soy grit
Recovery of protein from soy grit, its functional properties and possibility for the reduction of time of conven tional alkaline extraction by the assistance of enzymes were studied. Enzymatic treatment was performed by commercial preparations of cellulase (NS22086), xylanase (NS22083) and pectinase (Vinozym) (applied sepa rately or in combination) as well as by commercial carbohydrases cocktail (Enzyme complex, NS22119). Three different extractions were investigated - alkaline (at pH 8 for 1 h, 2 h or 3 h), enzyme-assisted aqueous (at pH 5.5 for 3 h) and enzyme-assisted alkaline extractions (enzymatic extraction for 1 h followed by alkaline extraction for 1 h or 2 h) at 50 � C and solid:liquid ratio 1:10 (w/v). The highest enhancement of recovery of protein was achieved by pretreatment of soy grit with enzyme cocktails. Treatment with Enzyme complex followed by 1 h alkaline extraction increased protein yield for 21% compared to 2 h alkaline extraction. Treatment by combi nation of individual cellulase, xylanase and pectinase followed by 2 h alkaline extraction enhanced protein yield for 13% in comparison to 3 h alkaline extraction. So, reduced time of alkaline extraction was attained by the assistance of carbohydrases cocktails with even positive effect on protein yield. In addition, protein from enzymeassisted alkaline extraction exhibited ameliorated solubility, emulsifying and whipping properties compared to alkaline extracted protein.
1. Introduction
sources such as extraction with aqueous and organic solvents (solutions with alkali, acid, salt, phenols, various buffers) that can be ultrasound, and enzyme-aided. Among all these methods, alkaline extraction is the most commonly used technique for protein extraction from plant and seed sources. Mechanism of alkali used for protein extraction from plants occurs through disrupting the cell wall by partial removal of lignin and by altering chemical composition and structure of (hemi) cellulose; in addition, the influence of alkali on protein extractability conducts also by its increased solubility (Sari et al., 2015). Great ad vantages of this traditional extraction method reflect in its simplicity, rapidity and low cost. Conventionally, soy protein isolates and concen trates are extracted from defatted soy flours using dilute alkali solutions (pH 8–9) at elevated temperature, followed with protein precipitation and concentration into a curd. Soy protein isolate yields are between 30 and 40% based on the defatted soy flakes/flour weight or approximately 60% of the protein in the flakes (Mounts et al., 1987). However, a traditional alkaline process can bring a few disadvantages as protein properties are changed along the extraction process. These changes include denaturation, racemization and formation of dehydro and
The importance of food proteins is well established - besides repre senting a source of energy and essential nutrients, these proteins fulfill another important role as they confer physicochemical characteristics that affect sensory properties and quality of food. Therefore proteins are considered the most valuable functional ingredients in food production. Over the last few decades growing global demand for food proteins has driven the search for new, sustainable protein sources based on plants able to potentially complement or replace animal proteins in various food applications (Rommi, 2016). Among them, soy occupies a promi nent place because soy protein is a complete protein that contains all essential amino acids required for normal human growth and develop ment (Singh et al., 2008). Nowadays, a consumption of soy products has increased significantly and the soybean protein is highly valued for its nutritional quality and functional properties. Soy food comes in dozens of forms including flour, protein isolates and concentrates, infant for €her et al., 2011). mulas and textured fibers (Stro A number of methods are used for the recovery of proteins from plant * Corresponding author. E-mail address: [email protected] (M.G. Antov).
https://doi.org/10.1016/j.jfoodeng.2019.109894 Received 10 June 2019; Received in revised form 20 December 2019; Accepted 22 December 2019 Available online 26 December 2019 0260-8774/© 2019 Elsevier Ltd. All rights reserved.
M.N. Perovi�c et al.
Journal of Food Engineering 276 (2020) 109894
cross-linked amino acids, reflected through poor solubility, lower nutritional quality and functional characteristics of extracted protein (Friedman, 1999; Sari et al., 2015). It was shown that these protein damaging processes were enhanced at higher pH and temperature and also increased with the increase in alkaline extraction time (Friedman, 1999; Schwass and Finley, 1984). Another approach to help disintegration of the plant cell wall that acts as a barrier for diffusion of protein into solvent, and to consequently enhance protein extractability, is the degradation of structural poly saccharides by the use of carbohydrases (Rosset et al., 2014). Generally, enzyme-assisted extractions are recognized as eco-friendly technologies as they offer a possibility of greener chemistry in a search for cleaner routes in food industry. Recent studies on enzyme-assisted extraction have shown faster extraction, higher recovery, reduced solvent usage and lower energy consumption when compared to non-enzymatic methods thus representing a potential alternative to conventional sol vent based extraction methods (Puri et al., 2012; Vergara-Barberan et al.,2015; Ribeiro Garcia De Figueiredo et al., 2018; V� asquez et al., 2019). Cell wall degrading carbohydrases - cellulase, pectinases and hemi cellulases are enzymes that hydrolyze plant cell wall components which leads to disruption of structural integrity and consequent increase in permeability of cell wall, finally resulting in enhancement of the extraction yield (Puri et al., 2012). Enzymes are usually used for the treatment of plant material prior to conventional methods of extraction. Some studies on enzymes-assisted extractions of soy flour showed promising results regarding improved yield of protein (Jung et al., 2006) and oil (Rosenthal et al., 2001) as well as nutritional and sensory properties of prepared extract (Wei et al., 2018). However, some of them reported no beneficial effect (Ouhida et al., 2002; Rosset et al., 2014) or even, under certain conditions, slightly decreasing effect (Rosenthal et al., 2001) on protein yield upon enzymatic treatment of soy flour. The aim of this study was to investigate the efficacy of cell wall degrading enzymes – cellulase, pectinase and xylanase, applied as single activity or in combination, in extraction of protein from defatted soy grit. The contents of cellulose, hemicellulose and pectin in soy flour/grit � and Zralý, cover a broad range of values (Jung et al., 2006; Písa�ríkova 2010; Rosenthal et al., 1998), therefore, the usage of enzymes that lead to increased protein extraction need to be established. The combination of enzymatic and alkaline extraction of protein with the aim of abridg ment of alkaline extraction by enzymatic pretreatment of soy grit was also investigated. In addition, functional characteristics of protein ob tained by enzymatic-alkaline extraction, such as solubility, emulsifying and whipping characteristics, important for its behaviour in food sys tems, were determined and compared to those of protein extracted by alkali.
Process, Novozymes) activity were used for enzymatically enhanced extraction of soy protein. In addition, one commercial cocktail of wide range of carbohydrase – cellulase, xylanase, pectinase, arabinanase and β-glucanase (Enzyme complex, NS22119, Novozymes’ Bioethanol Kit) was also applied for enzymatic treatment of soy grit. Cellulolytic activity of Cellulase complex and Enzyme complex assayed according to Ghose (1987) was determined to be 340 FPU/mL and 30 FPU/mL, respectively. One FPU was defined as the amount of enzyme that released 2 mg of reducing sugars from 50 mg of Whatman No.1 filter paper in 1 h under the standard conditions of hydrolysis (temperature 50 � C, pH 4.8). Xylanolytic activity of xylanase preparation NS22083 and Enzyme complex assayed according to Bailey et al. (1992) was determined to be 375 U/mL and 6 U/mL, respectively. One unit (U) of xylanase activity was defined as the amount of enzyme that liberates 1 mmol of xylose per minute under the assay conditions (temperature 50 � C, pH 5.3). Pectinolytic activity of Vinozym and Enzyme complex assayed ac cording to Patil and Dayanand (2006) was determined to be 7.8 U/mL and 1.1 U/mL, respectively. One unit (U) of pectinase activity was defined as the amount of enzyme that catalyzes liberation of 1 mmol of galacturonic acid per min at defined conditions of assay (temperature 45 � C, pH 4.5). 2.3. Extraction of protein from soy grit 2.3.1. Alkaline extraction Alkaline extraction of soy protein was carried out by distilled water with pH adjusted to 8 by NaOH, at solid:liquid ratio 1:10 (w/v) for 1 h, 2 h or 3 h, at 50 � C and constant stirring. After the extraction was completed, the suspension was centrifuged at 10,000 rpm for 15 min (Sorvall, RC-5B). Protein concentration in the supernatant was measured according to Lowry method (Lowry et al., 1951). 2.3.2. Enzymatic extractions In order to evaluate the effect of individual enzyme activities, extraction of soy protein was performed using single enzyme prepara tion in following dosages per gram of dry matter: 20 FPU cellulase from NS22086, 33 U xylanase from NS 22083 or 0.4 U pectinase from Vino zym. In addition, the effect of commercial enzyme cocktail (NS22119) and combination of individual enzyme preparation (NS22086, NS22083, Vinozym) named as Mix on recovery of protein was also evaluated. NS22119 was added at dosage corresponding to cellulolytic activity 20 FPU/g DM (followed by 4 U xylanase and 0.7 U pectinase per g DM), while carbohydrases Mix contained all three single enzyme dosages equal to those used in experiments with individual activities. Enzyme-assisted aqueous extractions were carried out under the same conditions as alkaline extraction - at 50 � C and solid:liquid ratio 1:10 (w/v) for 3 h but pH was 5.5. This pH fitted within the optimal pH ranges for all used enzyme activities (Novozymes, 2010). During the extraction, pH was controlled and adjusted to initial value with NaOH and HCl solutions. In order to inactivate enzymes after completed extraction, suspensions were boiled for 5 min at 100 � C. Control run was performed using same treatment conditions without enzymes. Combined enzymatic and alkaline extractions were performed by applying enzymatic treatment of defatted soy grit under the same con ditions described above for 1 h followed by alkaline extraction for 1 h or 2 h. After completed extraction processes, suspensions were cen trifugated at 10,000 rpm for 15 min (Sorval RC-5B) and protein con centration in supernatants was measured by Lowry method. Control was conducted at the same conditions described above but the part that corresponded to the enzymatic treatment was performed without enzymes.
2. Materials and methods 2.1. Soy grit Toasted soy grit, defatted by hexane extraction, was a gift from Sojaprotein (Be�cej, Serbia). Dry matter (DM) content determined by oven drying at 60 � C until constant mass was 98.7%. Protein content of defatted soy grit was determined by Kjeldahl (Official Methods of Analysis, 1995). Total dietary fibers (TDF), insoluble dietary fibers (IDF) and soluble dietary fibers (SDF) in defatted soy grit were determined using Total Dietary Fiber Assay Kit (Megazyme, Ireland). Before and during experimental analyses soy grit was stored in the refrigerator at constant temperature (4 � C) monitored by thermometer. 2.2. Commercial enzyme preparations
2.4. Protein precipitation and drying
Three commercial enzyme preparations having principally cellulo lytic (Cellulase complex, NS22086, Novozymes’ Bioethanol Kit), xyla nolytic (NS22083, Novozymes’ Bioethanol Kit) or pectinolytic (Vinozym
Before 2
electrophoresis
and
determination
of
functional
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Journal of Food Engineering 276 (2020) 109894
characteristics, extracts of soy protein were concentrated and dried. Knowing that isoelectric point of soy protein is at pH 4.5 (Jiang et al., 2010), 1M HCl was used to adjust pH in supernatants and precipitate extracted proteins. After precipitation, protein curds were collected by centrifugation (10,000 rpm, 10 min) and resolved in distilled water at pH 7. Obtained protein solutions were subjected to freeze drying at - 40 � C for 24 h at laboratory lyophilisation equipment (Martin Crist Alpha LSC 2–4, Osterode, Germany).
Table 1 Content of protein and dietary fibers in defatted soy grit. Component
Content (% DM)
Protein TDF IDF SDF
46.16 � 0.00 34.42 � 0.25 27.18 � 0.03 5.58 � 0.35
TDF ¼ total dietary fiber, IDF ¼ insoluble dietary fiber, SDF ¼ soluble dietary fiber.
2.5. Electrophoresis Samples from alkaline and combined enzymatic and alkaline ex tractions were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% TruPAGE Precast Gels (SIGMAALDRICH, Taufkirchen, Germany) under reducing conditions with discontinuous buffer system (Laemmli, 1970). Ten-μL samples (protein concentration 2 mg/mL) were loaded onto each well and electrophoresis was run at 180 V and 100 mA for 45 min. Gels were fixed and stained with Coomassie Blue R250 for 1 h and de-stained in distilled water, and the relative molecular weights of the protein fractions were compared with those of marker proteins (SERVA Electrophoresis, Heidelberg, Germany).
FSð%Þ ¼
3. Results and discussion In most grain legumes, which are used in human nutrition as valu able sources of protein, the content of cell wall materials i.e. dietary fiber ranges from 8% to 27.5% and differs among species and the vari eties; however, the content of dietary fiber in legume seed also varies according to their processing conditions (Guillon and Champ, 2002). Dietary fiber (DF) can be defined as carbohydrate polymers with 10 or more monomeric units which are not hydrolyzed by the endogenous enzymes in the small intestine of humans (CODEX Alimentarius Com mission, 2010). Dietary fiber consists of a variety of non-starch poly saccharides which include cellulose, hemicellulose, pectin, β-glucans and lignin. Hemicellulose, cellulose and lignin bind only little water and they are characterized as insoluble dietary fibers (IDF). On the other side, pectin substances and β-glucans are termed as soluble dietary fibers (SDF) because of their ability of binding water and swell. Defatted soy grit used in this study was analysed for protein and dietary fiber content and the results expressed as percentages of dry matter (DM) are pre sented in Table 1. Results showed that protein represented almost half of dry weight of defatted soy grit which generally, though somewhat higher, was in accordance with results reported in literature (Erickson, 2016; Rosen thal et al., 1998). Approximately one third of material dry weight was composed of cell wall components with dominant fraction of insoluble dietary fiber which can be attributed to high cellulose and hemicellulose content while soluble fiber related to pectin represented smaller portion. It is known that content of dietary fiber in soy flour/grit might cover a broad range of values. Literature data for TDF, IDF and SDF contents in different soybean genotypes ranged from 20.34 to 38.4 g, 18.19–35.4 g and 1.57–9.7 g per 100 g, respectively (Salmani et al., 2012; Pascoal et al., 2015; Písa�ríkov� a and Zralý, 2010). Determined contents of TDF, IDF and SDF in defatted soy grit used in this study were in good agreement with values reported previously.
(1)
Emulsion oil in water was prepared by mixing protein solution and sunflower oil in ratio 85:15 (v/v). Protein solutions were prepared by dissolving extracted protein in 0.3 M NaCl at pH 8 to make different concentrations – 0.2, 0.3 or 0.5 g/100 mL. Sodium azide was added as antimicrobial agent in concentration 0.02% (w/v). Oil and continuous water phase were homogenized with ULTRA-TURRAX (T-25, IKA) at 15,000 rpm for 10 min at 25 � C. Emulsion stability was evaluated by creaming test - immediately after preparation, emulsions were trans ferred into 10 mL graduated glass cylinders (VE); during the time emulsions separated into emulsion serum layer (VS) and emulsion cream; volume of serum layer was measured at room temperature and creaming index (CI) was calculated as: VS 100 VE
(2)
In addition, whipping ability of protein samples was investigated by determining foam capacity and foam stability. Protein solution (0.5 g/ 100 mL, 0.3 M NaCl, pH 8) was whipped at 15,000 rpm for 10 min at 25 � C. After whipping, the propeller was immediately removed and the glass cylinder sealed with parafilm to avoid the foam disruption. Vol umes were measured in starting minute (VO) and at 4th (V4) and 30th (V30) minute.Foam capacity (FC) was calculated as: FCð%Þ ¼
V4
V0 V0
100
(4)
Statistical analysis were carried out using Statistica (TIBCO Softver Inc.) to compare differences between results by the analysis of variance (One-way ANOVA), followed by Tukey’s HSD test. Results of statistical analysis were assumed to be significant when p < 0.05, very significant when p < 0.01 and highly significant when p < 0.001. Each experiment was carried out in triplicate and the results were presented herein as mean value � standard deviation.
2.7. Emulsifying and whipping characteristics
CIð%Þ ¼
100
2.8. Statistical analysis
Protein sample was dissolved in 0.3 M NaCl (Kinsella, 1979) at pH 8 to final concentration C1 of 4 g/L and thoroughly mixed on shaker at room temperature for 1 h after which pH was controlled and adjusted to 8 if necessary. Suspension was left overnight at 4 � C for better protein hydration and unsolved particles were separated by centrifugation at 10, 000 rpm for 10 min. Concentration of dissolved proteins in the super natant (C2), measured by Lowry method, was used to calculate solubility (S) according to Eq. (1): C2 100 C1
V0 V0
Microphotographs of prepared emulsions were taken on an optical microscope, Biooptica BEL3000, Germany at 40x magnification.
2.6. Solubility
Sð%Þ ¼
V30
3.1. Alkaline extraction
(3)
Defatted soy grit was extracted by alkaline aqueous solution (pH 8) at 50 � C for 1 h, 2 h or 3 h under constant stirring and after centrifu gation protein concentration in the supernatant was determined.
while foam stability (FS) was calculated according to Eq. (4): 3
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Journal of Food Engineering 276 (2020) 109894
3.2. Enzymatic extraction of protein Results presented in Table 1 showed that dietary fiber occupied almost one third of raw material dry weight, therefore in the next ex periments the effect of carbohydrases (individual or in combination) on the extraction of protein from soy grit was investigated. Dosages of in dividual activities were previously determined as those ones enabling the highest protein yield under applied conditions (data not shown). After solid/liquid separation, released protein was determined and re sults are presented in Fig. 2, along with results of the control extraction without enzymes. Action of cellulase, pectinase and Enzyme complex during aqueous extraction of defatted soy grit increased protein yield with high signif icance (p < 0.001) in comparison to control, while the addition of car bohydrases Mix increased it significantly (p ¼ 0.0364). However, when individual xylanase was added increase in protein yield was not signif icant (p ¼ 0.5265). Regarding the effect of individual enzyme activities, the most distinguished was that of cellulase (p ¼ 0.0001) with the protein yield of 27.56 � 1.24%, which can be explained by the high IDF i.e. cellulose content in soy grit. The highest protein yield 34.97 � 2.02% was achieved by commercial Enzyme complex representing highly sig nificant increase (p ¼ 0.0001) compared to control; this cocktail con tained wide range of carbohydrases including cellulase, pectinase and xylanase in the most adequate portions for the efficient joint hydrolysis of cell wall matrix and release of protein. The result of joint enzyme action within the Enzyme complex as the most efficient in the enhancement of protein yield was in agreement with recent model od plant cell wall as the single network composed from joined all structural polysaccharides (Dick-P�erez et al., 2011). In addition, it was shown that enzymatic degradation of one structural polysaccharide (cellulose) was significantly facilitated after removal of other cell wall components (Ouhida et al., 2002). It is worth to notice that protein yield reached by the use of commercial Enzyme complex was for 6% higher than that after 1 h alkaline extraction. However, comparing to the results of 3 h alkaline extraction protein yield from enzymatic aqueous extractions was lower from 16% to 57%. This can be explained by pH 5.5 of the
Fig. 1. Yield of protein from defatted soy grit obtained by alkaline extraction; conditions: pH 8, temperature 50 � C, extraction time 1 h, 2 h or 3 h. Different letters represent the differences in means of the samples, according to Tukey’s HSD test at p < 0.05 level.
Extraction yield of protein expressed as percentage of soy grit dry matter is presented in Fig. 1. Yield of protein significantly increased (p < 0.05) with increasing the time of alkaline extraction within investigated period. After 2 h extraction protein yield, 37.84 � 0.57%, was very significantly improved (p ¼ 0.0058) for 15% compared to yield from 1 h extraction. Further prolongation to 3 h extraction enabled achievement of protein yield 41.03 � 1.96% which represented significant increase compared to 2 h extraction (p ¼ 0.0398). This result was in accordance with yield of protein extracted from defatted soy flour by alkali under similar con ditions (Mounts et al., 1987; Sari et al., 2015).
Fig. 2. Yield of protein from defatted soy grit ob tained by enzyme-assisted aqueous extractions; con ditions: pH 5.5, temperature 50 � C, time: 3 h; enzyme dosages/g DM: 0.4 U pectinase (Vinozym), 33 U xylanase (NS22083); 20 FPU cellulase (NS22086); E. complex - 0.7 U pectinase, 4 U xylanase and 20 FPU cellulase; MIX - 0.4 U pectinase (from Vinozym), 33 U xylanase (from NS22083) and 20 FPU cellulase (from NS22086); control without enzymes. Different letters represent the differences in means of the samples, according to Tukey’s HSD test at p < 0.05 level.
Enzymatic extraction
50 45
a
Extraction yield (% DM)
40 35
b
b
30 25 20
c
c,d
d
15 10 5 0
control
pectinase
xylanase
cellulase
E complex 4
MIX
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Journal of Food Engineering 276 (2020) 109894
Fig. 3. Yield of protein from defatted soy grit ob tained by enzyme-assisted alkaline and alkaline ex tractions: a) 1 h at pH 5.5 followed by 1 h pH 8, and b) 1 h at pH 5.5 followed by 2 h at pH 8; temperature: 50 � C; enzymes dosages/g DM: 0.4 U pectinase (Vinozym), 33 U xylanase (NS22083); 20 FPU cellu lase (NS22086); E. complex - 0.7 U pectinase, 4 U xylanase and 20 FPU cellulase; MIX - 0.4 U pectinase (from Vinozym), 33 U xylanase (from NS22083) and 20 FPU cellulase (from NS22086); controls: 1 h at pH 5.5 without enzymes followed by 1 h or 2 h at pH 8 and 50 � C. Different letters represent the differences in means of the samples, according to Tukey’s HSD test at p < 0.05 level.
a)
b)
extraction experiments, which was appropriate for the enzyme action but did not enable maximal protein solubility as discussed previously (Sari et al., 2015). Presented protein yield corresponded to this extracted under similar experimental conditions from soy flakes (Jung et al., 2006).
regardless Enzyme complex (increase for 38%) or carbohydrases Mix (increase for 22%) was used for enzymatic treatment followed by 1 h alkaline extraction (Fig. 3a). When treatment with Enzyme complex and Mix was followed by 2 h alkaline extraction protein yield was increased for 31% and 28%, respectively, which also represented highly signifi cant improvement (p ¼ 0.0002) compared to corresponding control (Fig. 3b). Moreover, after enzymatic treatment with carbohydrases Mix and Enzyme complex followed by 1 h alkaline extraction protein yield, 40.87 � 1.37% and 45.93%, respectively, was almost equal or higher than this after 3 h alkaline extraction (41.03 � 1.97%). On the other side, statistical analysis showed that the enhancement of protein yield by treatment with individual enzymes was significant only when it was followed by 1 h alkaline extraction. Observed enhancement was at very significant level (p ¼ 0.0014) and significant level (p ¼ 0.0234) for xylanase and pectinase, respectively. So, results showed that under applied conditions treatment with enzyme cocktails enhanced protein extraction more markedly than that with individual enzymes which was contrary to findings of Jung et al. (2006). Nevetheless, in comparison to 2 h alkaline extraction, protein yields
3.3. Enzyme-assisted alkaline extraction Combination of enzyme-assisted aqueous extraction and alkaline extraction was performed primarily to reduce alkaline extraction time in order to decrease possible protein damage and formation of undesired products. Moreover, this combined extraction process should overcome restraints of enzyme-assisted aqueous extraction regarding the effect of applied pH on protein solubility. Two combined extractions included 1 h enzymatic treatment followed by 1 h or 2 h alkaline extraction, and results of protein yield as the first response are presented in Fig. 3. In two and 3 h combined extractions the most prominent increase of protein yield in comparison to corresponding controls was achieved with enzyme cocktails. This increase was highly significant (p ¼ 0.0002) 5
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Journal of Food Engineering 276 (2020) 109894
Table 2 Functional characteristics of protein from defatted soy grit extracted by alkaline and carbohydrases MIX-assisted alkaline extraction. Extractiona
Solubility (%)
Foam capacity (%)
Foam stability (%)
Alkaline
73.67 � 1.01a 79.90 � 0.80b
57.50 � 3.53a
49,00 � 1.41a
107,00 � 4.24b
89.73 � 0.37b
Carbohydrases MIXassisted a
Alkaline: 3 h at pH 8 and 50 � C; carbohydrases MIX-assisted: 0.4 U pectinase, 33 U xylanase and 20 FPU cellulase activity/g DM, 1 h at pH 5.5 followed by 1 h alkaline extraction at pH 8, 50 � C. Different letters represent the differences in means of the samples, according to Tukey’s HSD test at level p < 0.001 for solubility and foam stability, and at level p < 0.01 for foam capacity.
order to perceive the effect of applied extraction conditions. Analysed protein samples originated from extractions yielding approximately equal amounts of protein. 3.5.1. Solubility It is well known that solubility of food proteins is one of most valu able functional properties in general. Typically, higher protein solubility correlates with good gelling, foaming and emulsifying properties (Joo kyeing, 2011). Results of solubility of the investigated protein samples at pH 8 are presented in Table 2. The higher solubility, 79.90 � 0.80%, was determined for protein extracted by combined treatment with carbo hydrase MIX; though lower than literature data for soy protein isolate (Rickert et al., 2004) this represented highly significant improvement (p ¼ 0.0004) of solubility in comparison to sample from alkaline extraction. It is known that protein properties are affected by the processing treatments and environment (Kinsella, 1979) and extraction conditions that include heat and alkaline treatment can be related to denaturation leading to lower solubility (Sari et al., 2015). Reason for the enhanced solubility of protein from enzyme-assisted alkaline extraction can be related with lower degradation of protein during extraction process due to reduced duration of protein exposure to alkali.
Fig. 4. SDS–PAGE of proteins from pectinase-assisted followed by 2 h alkaline (line 1), 3 h alkaline (line 2) and carbohydrase MIX-assisted followed by 2 h alkaline (line 3) extractions, loaded by 20 μg each; protein molecular weight markers are in lane M and corresponding molecular weights (in kDa) are indicated at side.
after treatment with individual enzymes followed by 1 h alkaline extraction had mutually very close values. Statistical analysis of the results obtained from enzyme-assisted al kali extractions showed that prolongation of alkaline part of extraction was not justified for all enzymatic treatments (individual or in combi nation). Judging by the significance of the enhancement, 2 h compared to 1 h alkaline extraction after enzymatic treatment improved protein extractability only when cellulase (significantly, p ¼ 0.012) and carbo hydrases Mix (highly significantly, p ¼ 0.0008) were used. With respect to protein yield presented results showed that by enzymatic treatment of soy grit particularly with enzyme cocktails reduction of time of alkaline extraction can be accomplished, with no decreasing but even with the enhancing effect.
3.5.2. Emulsifying characteristics The ability of protein to help the formation and stabilization of emulsions is very important for many food applications. The stability of emulsions prepared with protein samples from alkaline and carbohy drases Mix-assisted extractions was evaluated by monitoring of the creaming index in emulsions prepared with 15% oil and values of this indicator are shown in Fig. 5. Results revealed that increase in protein concentration of both tested samples increased the stability of prepared emulsions and that for both of them final values of the creaming index were similar. Comparing stabilities of emulsions prepared with equal concentration of protein extracted by different procedures, differences between them increased also with increasing protein concentration. As a result, the highest applied concentration of protein from enzyme-assisted alkaline extrac tion prolonged time needed for emulsion separation for approx. 10 min in comparison with alkali extracted protein under applied conditions thus indicating its improved emulsifying activity regarding stabilizing effect on oil-in-water emulsions. It is known that surface activity of protein that is related to their ability to lower the interfacial tension between water and oil is closely correlated with solubility and flexibility of protein molecule (Kinsella, 1979). In addition, it was shown that although denatured soy protein expressed higher interfacial pressure than native, emulsions prepared with native sample were more stable (Palazolo et al., 2003). So, presented results can be explained by milder extraction conditions in combined extraction comprised from enzymatic treatment and reduced exposure to alkali, which enabled higher solu bility and possibly higher ability for structure restoration (i.e. flexibility) of protein leading to improved stabilizing effect on emulsions. This
3.4. SDS-PAGE Samples from alkaline as well as from enzyme-assisted alkaline extraction were subjected to electrophoresis to evaluate pattern of extracted protein. After isoelectric precipitation and freeze-drying, samples of soy protein obtained by alkaline and two combined extrac tions were loaded in equal protein amounts. Proteins extracted by combination of enzymatic treatment with carbohydrases MIX and Vinozym followed by 2 h alkaline extraction were chosen based on the yield higher and lower than this from alkaline extraction, respectively. Results from SDS-PAGE (Fig. 4) showed that protein pattern in all ana lysed samples was unique indicating the presence of subunits of major soy storage proteins with approximate molecular weights 80, 75, 50, 40 and 20 kDa (Krishnan et al., 2009; Rovaris et al., 2013; Thanh and Shibasaki, 1977) as well as around 17 kDa band corresponding to Kunitz ~o �n, 1995). This revealed that the trypsin inhibitor (Petruccelli and An same protein fractions were extracted by alkaline and enzyme-assisted alkaline extractions indicating that differences in yields can be attrib uted to variation in degree of cell wall destruction which led to differ ences in protein release. 3.5. Functional characteristics of extracted protein Some functional characteristics of protein samples from alkaline and enzyme-assisted alkaline extractions were determined and compared in 6
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Journal of Food Engineering 276 (2020) 109894
Fig. 5. Creaming index of emulsions prepared with protein from a) 3 h alkaline and b) carbohydrases MIX-assisted alkaline extractions.
result is also in an agreement with higher solubility expressed by protein originated from enzyme-assisted alkaline in comparison to that from alkaline extraction. Emulsifying ability of protein samples was also perceived through analysis of images of emulsions previously tested for stability (Fig. 6).
Practically, images confirmed previous finding based on the measure ment of creaming index considering the fact that emulsions with smaller droplets size are generally more stable. It was visible that protein from both samples at higher concentrations produced smaller oil drops; generally, it is known that higher protein concentration is able to better 7
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Journal of Food Engineering 276 (2020) 109894
Protein concentration (%, w/v)
Extraction
0.2
0.3
0.5
Alkaline
MIX-assisted alkaline
Fig. 6. Images of oil droplets from emulsions with different concentrations of protein from alkaline and carbohydrases MIX-assisted alkaline extractions.
cover surface area of oil drops and to stop progressive coalescence of oil droplets and oil separation (Palazolo et al., 2003). Furthermore, it was also visible that protein from enzyme-assisted alkaline extraction pro duced relatively smaller oil droplets. Another property of protein that is important for some food appli cations is the capacity to form stable foams with gas by forming impervious protein films. Whipping ability of two protein samples was evaluated by measuring foam capacity and stability (Table 2). Higher foam capacity and stability, 57.5 � 3.53 and 107 � 4.24%, respectively were determined with protein from carbohydrases Mix-assisted alkaline in comparison to that from alkaline extraction (49 � 1.41 and 89.73 � 0.37%, respectively). This improvement was at level very significant (p ¼ 0.0064) and highly significant (p ¼ 0.0009) for foam capacity and foam stability, respectively. Good whipping characteristics of proteins are usually related to their good solubility in disperse (aqueous) phase but also to greater exposure of hydrophobic regions caused by dena turation (Kinsella, 1979; Van Vliet et al., 2002). However, it was also shown that soy protein exhibited superior foaming properties when it was native and soluble (Yasumatsu et al., 2014). Our results showed that reduced time of the exposure to alkali by previous enzymatic treatment favored extraction of protein with improved whipping characteristics indicating its increased ability of making film at the interface, which was in agreement with previously discussed results of its higher solubility.
treatment with commercial Enzyme complex and carbohydrases Mix (cellulase, xylanase and pectinase) followed by alkaline extraction. Combination of enzymes appeared to be the most adequate for the efficient hydrolysis of cell wall matrix and release of protein. Beside the positive effect on protein yield, functional characteristics of protein (solubility, emulsifying and whipping properties) from enzyme-assisted alkaline extraction were advanced compared to those from alkaline extraction. Presented results allowed reduction of time of alkaline extraction by its combination with enzymatic treatment, which posi tively affected both protein yield and functional properties as desirable attributes for protein application in wide range of food products. CRediT authorship contribution statement Milica N. Perovic�: Formal analysis, Conceptualization, Writing original draft. Zorica D. Kne� zevi� c Jugovi� c: Project administration, Validation. Mirjana G. Antov: Conceptualization, Supervision, Writing - review & editing. Acknowledgement The financial support from EUREKA Project SOYZYME (Grant No E! 9936) is greatly acknowledged. The authors also acknowledge Novo zymes for kindly gifted us by Novozymes Cellulosic Ethanol Enzyme Kit.
4. Conclusion
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